TRAF7 is an essential regulator of blood vessel integrity during mouse embryonic and neonatal development

Summary Targeted deletion of TRAF7 revealed that it is a crucial part of shear stress-responsive MEKK3-MEK5-ERK5 signaling pathway induced in endothelial cells by blood flow. Similar to Mekk3-, Mek5- or Erk5-deficient mice, Traf7-deficient embryos died in utero around midgestation due to impaired endothelium integrity. They displayed significantly lower expression of transcription factor Klf2, an essential regulator of vascular hemodynamic forces downstream of the MEKK3-MEK-ERK5 signaling pathway. In addition, deletion of Traf7 in endothelial cells of postnatal mice was associated with severe cerebral hemorrhage. Here, we show that besides MEKK3 and MEK5, TRAF7 associates with a planar cell polarity protein SCRIB. SCRIB binds with an N-terminal region of TRAF7, while MEKK3 associates with the C-terminal WD40 domain. Downregulation of TRAF7 as well as SCRIB inhibited fluid shear stress-induced phosphorylation of ERK5 in cultured endothelial cells. These findings suggest that TRAF7 and SCRIB may comprise an upstream part of the MEKK3-MEK5-ERK5 signaling pathway.

In addition to MEKK3 and MEK5, TRAF7 binds SCRIB TRAF7 and SCRIB participate in ERK5 phosphorylation in endothelial cells

INTRODUCTION
The TRAF (TNF receptor-associated factor) family consists of seven adaptor proteins with diverse roles in intracellular signaling. 1 While TRAF1 to TRAF6 are well-characterized molecules involved in immunity and inflammation, TRAF7 function remains unclear. TRAF7 was shown to be mutated in 30% of meningiomas, the most common primary tumor of the central nervous system (CNS). [2][3][4] In addition, de novo mutations in Traf7 gene were described in patients with developmental delay, congenital heart defects, limb and digital anomalies, and dysmorphic features. 5,6 All reported somatic as well as germline Traf7 mutations are hemizygous missense and fall into different positions within the C-terminal part of the protein, suggesting gainof-function and/or dominant-negative effects of the resulting mutant protein, rather than loss of function resulting in haploinsufficiency. Although almost all identified mutations are recurrent (the same in different tumors or patients), somatic and germline mutations do not overlap. For example, while a germline R655Q mutation appears in almost 30% of patients with TRAF7 syndrome, 5 no published meningiomas have a mutated R655, implying that signaling programs initiated by somatic versus germline TRAF7 variants may be different and may direct significantly divergent courses of action.
All mammalian TRAFs, with the exception of TRAF1, contain a characteristic N-terminal RING (really interesting new gene) finger domain, several adjacent zinc fingers (ZFs), and a coiled coil (CC) motif, which fosters formation of TRAF trimers. 1 At the C terminus, six TRAFs (1 through 6) carry a conserved TRAF domain responsible for binding to other proteins, including cell surface receptors and intracellular adapters. 7 Unlike the rest of the family members, TRAF7 lacks the TRAF domain and instead contains a tryptophan-aspartic acid (W-D) dipeptide (WD40) domain consisting of seven WD40 repeats. For that reason, TRAF7 is often placed into a RING finger and WD domain (RFWD) family of proteins and is called RFWD1. This group also includes RFWD2, an E3 ubiquitin ligase constitutive photomorphogenic 1 (COP1), 8,9 and RFWD3. 10 Importantly, the structure of COP1 and RFWD3 RING fingers is very different from that of the canonical TRAF-family RING domain of TRAF7. Thus, it is not surprising that a recent phylogenetic analysis assigned TRAF7 into a separate group that developed independently from other TRAFs throughout vertebrate evolution. 11 In fact, unlike other TRAFs, TRAF7 remained highly conserved during evolution; humans and lampreys (Petromyzon marinus) share more than 80% of the whole protein and 93% of the WD40 domain amino acid (aa) sequences. Thus, TRAF7 is a unique protein without close paralogs in vertebrates.
Over the years, TRAF7 has been shown to participate in different signaling pathways, ranging from activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) to Activator protein 1 (AP-1), 12 and to suppress endothelial cell hyperpermeability in inflammation. 13 Two decades ago TRAF7 was discovered as a part of a mitogen-activated protein kinase kinase kinase 3 (MAP3K3, also known as MEKK3)-associated protein complex using tandem affinity purification. 12 This complex also included dual specificity mitogen-activated protein kinase kinase 5 (MAP2K5, also known as MEK5), serine/threonine-protein kinase MARK2 (also known as EMK1), prefoldin 2 (PFDN2), heat shock protein 70 (HSP70), and p53 and DNA damage regulated 1 (PDRG1, also known as C20orf126). 12 In the same year, Xu et al. independently confirmed the association of TRAF7 with MEKK3 14 and suggested that TRAF7 is important for MEKK3-mediated activation of AP-1 and DNA damage-inducible transcript 3 protein (DDIT3, also known as CHOP). TRAF7 was also shown to associate with a number of other molecules, including cylindromatosis 1 (CYLD1), 15 transcriptional activator c-MYB, 16 myoblast determination protein 1 (MYOD1), 17 NF-kappa-B essential modulator (NEMO), 18 and roundabout homolog 4 (ROBO4). 13 A recent high-throughput affinity-purification mass spectrometry interactome study demonstrated that TRAF7 interacts with MEK5 and indirectly with MEKK3. 19 In addition, TRAF7, together with cerebral cavernous malformations 1 (CCM1, also known as KRIT1) and CCM2, precipitated with MEKK3, which was necessary and sufficient for CCM-dependent expression of the shear stress-responsive transcription factors Krü ppel-like factor 2 (KLF2) and its close paralog KLF4. 20 MAPK signaling pathways are activated by a wide variety of stimuli such as mitogens, osmotic stress, and pro-inflammatory cytokines. Each MAPK cascade consists of at least three enzymes that are activated in chains following phosphorylation: a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAP kinase (MAPK). MEKK2 and MEKK3 are unique among MAP3K kinases because each of them possesses an N-terminal PB1 (Phox and Bem1) domain, which is used to bind to PB1 domain of MEK5. 21 Following hetero-dimerization with MEK5, MEKK2 or MEKK3 phosphorylates and activates MEK5 in response to different stimuli, including blood flow shear stress. 21 In turn, activated MEK5 phosphorylates and activates extracellular signal-regulated kinases 5 (ERK5, also known as MAPK7 and Big MAPK Kinase 1 [BMK1]), which was originally identified as a MAPK activated by both osmotic and oxidative stress. 22 Interestingly, ERK5 is also unique among MAPKs due to the presence of a C-terminal extension, which gives the protein approximately twice the molecular weight of other MAPKs. The MEK5/ERK5 pathway is known to be activated by shear stress and to sustain resistance of endothelial cells to apoptosis. 23,24 This pathway is also important for shear stress-dependent induction of KLF2. [25][26][27][28] Despite significant progress in understanding of the role of MEKK3-MEK5-ERK5-KLF2 pathway in endothelial cells, the upstream mechanism of shear-stress signal transduction is still unclear. Here, we generated Traf7-deficient mice to elucidate the physiological role of TRAF7. Global Traf7 deletion resulted in embryonic lethality. The Traf7 À/À embryos failed to develop beyond E10 due to impaired blood vessel integrity. A detailed pathology analysis revealed evidence of intraembryonic hemorrhage and hypoxia. Transcriptome analysis of failing embryos demonstrated significantly low expression of transcription factor KLF2 among other genes. We also show that besides MEKK3 and MEK5 TRAF7 associates with a planar cell polarity protein scribble homolog (SCRIB) and that downregulation of TRAF7 as well as SCRIB inhibited fluid shear stress-induced phosphorylation of ERK5. Thus, TRAF7 is a part of MEKK3-MEKK5-ERK5 signaling pathway.

Targeted deletion of Traf7 gene causes embryonic lethality
To investigate the function of TRAF7 in vivo, we generated mice with a conditionally targeted Traf7 allele. The conditional allele contains loxP sites flanking exons 2 and 14, which include coding sequence for the first 373 amino acids of the protein through the middle of the first WD40 repeat ( Figure S1A). Mice homozygous for the Traf7-floxed allele (Traf fl/fl ) were healthy and fertile. To produce whole-body, global deletion of Traf7 mice (Traf7 GLOko = global knockout), we crossed Traf7-flox mice with E2a-Cre-trangenic animals, which carry a Cre transgene under the control of the adenovirus EIIa promoter that targets expression of Cre recombinase ubiquitously to cells of the early mouse embryo. 29 Heterozygous (Traf7 GLOhet ) mice were phenotypically normal and fertile. Genotyping of Traf7 GLOhet intercrosses revealed that no Traf7 GLOko pups were born ( Figure 1A). The statistical analysis was performed to examine if there is deviation from ll OPEN ACCESS expected Mendelian inheritance outcomes of the alleles during embryogenesis. The relation between these variables was not statistically significant for embryos on days E9.5 (X 2 (2, N = 181) = 1.98, p = 0.3716) and E10.5 (X 2 (2, N = 94) = 1.66, p = 0.4360). The deviation of Traf7 GLOhet progeny from Mendelian distribution of alleles was statistically significant for embryos at E11.5 (X 2 (2, N = 17) = 10.72, p = 0.0047) and very statistically significant for newborns (X 2 (2, N = 352) = 33.44, p = 0.0001). Therefore, analysis of timed mating revealed normal Mendelian segregation ratios for the disrupted Traf7 alleles until embryonic day 10.5 (E10.5). iScience Article All E9.5 Traf7 GLOko embryos were viable, as determined by the presence of visible heartbeat (Videos S1, S2, S3, S4, and, S5). The Traf7 GLOko embryos and yolk sacs displayed normal appearance and no differences from wild-type (WT) littermates ( Figure 1B, top and middle panel). After approximately E10, the mutant embryos displayed developmental delay and appeared smaller and paler than WT embryos; mutants also showed evidence of cerebral hemorrhage by E10.5 ( Figure 1B, bottom panel). This phase in embryonic development coincides with the onset of embryonic blood flow and the establishment of a fully functional circulation, 30 suggesting that TRAF7 may be important for the integrity of blood vessels in developing embryos. Because the phenotype of Traf7 GLOko embryos was similar to phenotypes observed in Mekk3-, Mek5-and Erk5-deficient embryos, which likewise died in utero around E10, 31-35 we examined the expression of these proteins in Traf7 GLOko embryos. As shown in Figure S1D, there was no expression of TRAF7 in E9.5 Traf7 GLOko embryos, while expression of MEKK3, MEK5, and ERK5 remained normal, indicating that the expression levels of MEKK3, MEK5, and ERK5 were not altered by removal of TRAF7, and the lethality of Traf7 GLOko embryos was indeed associated with complete TRAF7 deficiency.
Because the MEKK3-MEK5-ERK5 signaling cascade is essential for embryonic vascular integrity and cardiovascular development, 36,37 we next investigated the expression of the endothelial cell marker platelet endothelial cell adhesion molecule 1 (PECAM-1, also known as CD31), in Traf7 GLOko embryos. There were no obvious visual differences in the appearance of blood vessel networks between WT and Traf7 GLOko embryos at E9.5 ( Figure 1C, top panel). At E10.5, the blood vessels of Traf7 GLOko embryos appeared discontinuous and less branched compared to blood vessels in WT embryos ( Figure 1C, middle panel). Quantification of intersomitic vessel structure using Angiogenesis Analyzer for ImageJ, which was developed for the analysis of in vitro angiogenesis patterns 38 and later adopted for in vivo vascular changes in retina, 39 demonstrated that blood vessels of E10.5 Traf7 GLOko embryos were significantly more fragmented compared to WT controls. There was lower numbers of junctions, segments, and branches per square unit. Consistently, the number of isolated segments was higher, although not significantly, in Traf7-GLOko embryos ( Figure 1D). Thus, the initial vascularization occurs normally in Traf7 GLOko embryos, but the subsequent structural integrity of blood vessels was compromised, similarly to Mekk3-, Mek5-and Erk5deficient embryos. 31-35 These findings imply that TRAF7 may contribute to MEKK3-MEK5-ERK5 signaling pathway during embryonic development.

Traf7-deficient embryos display abnormal heart development
Although, histological analysis of E9.5 embryos revealed that Traf7 GLOko embryos had normal morphology (Figure 2A), the pericardium was broken in all Traf7 GLOko embryos we examined, probably due to lack of elasticity or excessive effusion. It is also possible that disruption of the pericardium happened during embryo handling, but we did not encounter a single broken pericardium in any of our WT embryos. Hence, E9.5 Traf7 GLOko hearts appeared to have a significantly different shape and protruded further than WT hearts (Figure 2A). A closer iScience Article examination demonstrated grossly normal heart looping in Traf7 GLOko embryos with the atrioventricular canal extending more forward compared to WT controls. E9.5 Traf7 GLOko embryos also displayed an abnormal ventricle position accompanied by a higher deposition of cardiac jelly in endocardial cushions compared to WT counterparts. This phenotype is consistent with impaired MEKK3 signaling and low endocardium expression of KLF2, which was shown to control cardiac jelly production. 20,40 At E10.5, Traf7 GLOko embryos displayed signs of tissue resorption and appeared more fragile than WT controls ( Figure 2B). In contrast to WT hearts, which grew in size and developed interventricular foramen dividing the two ventricles, E10.5 Traf7 GLOko hearts did not increase in size and showed significant shrinkage of heart chambers with manifestations of degradation in surrounding tissue. Moreover, all experimental E10.5 Traf7 GLOko embryos without exception had broken heart walls and no blood cells in the heart cavities compared to their WT counterparts, indicating irreversible vascular failure by E10.5.

Altered gene expression associated with TRAF7 deficiency in mouse embryo
To understand gene expression differences between E9.5 WT and Traf7 GLOko embryos, we compared their transcriptome profiles by RNA sequencing (RNA-seq). The analysis revealed that six WT embryos formed a tight cluster following principal-component analysis (PCA) ( Figure S2A). Although six Traf7 GLOko embryos distributed more widely, three of them were in close proximity to the WT cluster, suggesting a recent branching of WT and Traf7 GLOko transcriptional profiles. Indeed, the majority of identified genes (11,661) iScience Article were common between these two groups ( Figure 3A), and only 275 and 575 of differentially expressed genes (DEGs) were specifically expressed in either WT or Traf7 GLOko embryos, respectively. Significant DEGs were defined as those that had both an absolute log2 fold change R1 and a false discovery rate (FDR)-adjusted p value %0.05 for each comparison independently. Hierarchical clustering based on gene expression profiles revealed high similarity of expression patterns between samples within each group and substantial differences between them ( Figure S2B).
Gene set enrichment analysis in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database demonstrated that WT and Traf7 GLOko embryos were different in the expression of many gene targets associated with the hypoxia-inducible factor 1 (HIF-1) signaling pathway ( Figure 3B), suggesting embryonic distress resulting from insufficient blood flow in the absence of TRAF7. Detailed analysis of specific genes on a volcano plot demonstrated that Traf7 GLOko embryos were missing Traf7, as expected ( Figure 3C). The most prominent downregulated DEG was the zinc-finger transcription factor Klf2, a key flow-responsive gene in endothelial cells. 41 Interestingly, WT embryos also expressed approximately 1.5-fold higher levels of the Klf2 homolog Klf4 than Traf7 GLOko embryos, but without statistical significance (Table S1). Next, we validated the downregulated expression of Klf2 in Traf7 GLOko embryos by quantitative RT-PCR ( Figure S2C). Importantly, the expression of Cd31 was the same in both types of embryos, suggesting a comparable number of WT and Traf7 GLOko endothelial cells. Because Klf2 is almost exclusively expressed in the endothelium during mouse embryonic development, and because it was shown to be selectively induced by fluid shear forces 26,27,42 or biomechanical stimuli via the MEK5-ERK5 pathway, 28,42 our findings suggest an impaired MEKK3-MEK5-ERK5-KLF2 signaling cascade in Traf7 GLOko embryos.
The DEG with the highest level of expression in WT embryos compared to Traf7 GLOko embryos was AU021092 ( Figure 3C), a gene of unknown function. Because this gene is expressed by cardiomyocytes in E8.5 embryos, 43 these results imply an abnormal heart function in Traf7 GLOko embryos. The other two DEGs downregulated in Traf7 GLOko embryos were Serpina6, also known as transcortin, and Nppc, also known as C-type natriuretic peptide (CNP). While transcortin is a major corticosteroid transporter in the blood, NPPC is a major heart-protective natriuretic peptide upregulated by shear stress in endothelial cells. 44,45 One of the genes that was increased in Traf7 GLOko embryos, compared to WT counterparts, was BCL2/adenovirus E1B interacting protein 3 (Bnip3), a pro-apoptotic gene induced by hypoxia. [46][47][48] Interestingly, Erk5 À/À embryos also displayed low expression of Klf2 and high expression of Bnip3 when compared to WT controls. 25 Taken together, these results once again suggest that TRAF7 is an integral part of vascular integrity regulated by MEKK3-MEK5-ERK5-KLF2 pathway in embryonic development.

Endothelial-specific deletion of Traf7 confers embryonic lethality
To examine whether endothelium-specific expression of TRAF7 is required during embryonic development, we crossed Traf7-floxed mice with Tie2-Cre + animals. 49 The Tie2-Cre transgene drives Cre expression throughout early endothelium and in hematopoietic cells, but not in smooth or cardiac muscle. 50 The intercross produced no Traf7 fl/fl ;Tie2-Cre + pups (Traf7 ECko = endothelial cell knockout), indicating that conditional deletion of Traf7 in endothelial cells leads to embryonic lethality ( Figure 4A). Analysis of timed mating revealed ratios of progeny were non-Mendelian; specifically, the observed proportion of Traf7 ECko progeny (0%) differed from expected 25% (X 2 (3, N = 81) = 27, p < 0.0001). Interestingly, the proportion of Traf7 EChet newborn pups (22.2%) was also lower than the expected 33% of remaining pups after Traf7 ECko embryonic lethality; X 2 (2, N = 81) = 5, p = 0.03), suggesting that a single copy of Traf7 in some embryos was not sufficient for survival during gestation. As with Traf7 GLOko embryos, E9.5 Traf7 ECko embryos were indistinguishable from Traf7 fl/fl (control) littermates, while E10.5 Traf7 ECko embryos appeared to be smaller and underdeveloped compared to controls ( Figure 4B). In addition, some Traf7 ECko embryos were bloodless and/or had visible hemorrhages. Vascular development of E9.5 embryos was assessed by staining with anti-CD31 antibody and looked intact in both Traf7 EChet and Traf7 ECko embryos ( Figure 4C). In contrast, E10.5 Traf7 ECko embryos displayed fragile blood vessels with interrupted connections in the head and in the back of the trunk where intersomitic arteries fuse to dorsal longitudinal anastomotical vessels.
Quantification of intersomitic vessel structure demonstrated that blood vessels of E10.5 Traf7 ECko embryos were significantly more fragmented compared to Traf7 EChet controls. There were significantly lower numbers of junctions, segments, and branches, while higher numbers of isolated segments inTraf7 GLOko embryos ( Figure S3). Overall, it appears that Traf7 ECko embryos die around E10, which is similar to the timing of death of Traf7 GLOko embryos. Taken together, these results indicate that TRAF7 is essential for normal endothelium function during embryonic development. iScience Article To gain insight into the genes regulated by TRAF7 in embryonic endothelial cells, we performed RNA-seq analysis of Traf7 ECko embryos at E9.5. Traf7 EChet embryos were chosen as a control group to cancel out any interference resulting from the presence of the Tie2-Cre transgene. Thus, both of these groups of embryos iScience Article only differ by a single copy of Traf7 in any given cell after Cre-dependent deletion. The analysis revealed that Traf7 EChet embryos formed a relatively tight cluster following PCA ( Figure S4A), while Traf7 ECko embryos failed to form a compact cluster. Three Traf7 ECko embryos were in close proximity to the control cluster, suggesting incomplete deviation of the two transcriptional profiles, probably due to inefficient deletion of the floxed Traf7 gene. Indeed, the vast majority of identified genes (11,787) were common between the two groups, and only 172 and 218 of the DEGs were upregulated in control and Traf7 ECko embryos, respectively ( Figure S4B). Nevertheless, hierarchical clustering of transcriptome profiles revealed high sample similarity within each group and a good separation between groups ( Figure S4C). Gene set enrichment analysis in the KEGG pathway database unveiled enrichment of cellular senescence, AMP-activated protein kinase (AMPK), and HIF-1 signaling in Traf7 ECko embryos compared to Traf7 EChet (Figure 4D), suggesting a deviation in energy balance between the two groups due to Traf7 deficiency.
In agreement with transcriptome analysis of Traf7 ECko embryos, Klf2 turned up to be the most statistically significant DEG with lower expression in embryos with endothelial-specific Traf7 deletion compared to the controls ( Figure 4E and Table S2). Downregulation of Klf2 in Traf7 ECko embryos was confirmed with quantitative RT-PCR ( Figure S4D). There was also decreased expression of the nitrogen oxide-transporting transmembrane pore aquaporin-1 (Aqp1) previously identified as a direct subject to KLF2-mediated positive regulation in endothelial cells, 51 suggesting dysregulated KLF2 activity and impaired endothelial cell function in Traf7 ECko embryos. Out of broadly expressed genes, Traf7 ECko embryos demonstrated lower expression of mt-Nd6, mt-Co1, mt-Nd4, and mt-CytB, subunits of the mitochondrial membrane respiratory chain enzymes, indicating attenuated aerobic respiration in tissues of Traf7 ECko embryos. Consistently, galactokinase Galk1 and mitochondrial Ndufa4l2, two direct targets of HIF-1, 52,53 were among the most statistically significant DEGs highly expressed in Traf7 ECko embryos compared to Traf7 EChet counterparts ( Figure 4E). These results are in agreement with recent observation that the mitochondrial pathway participates in the expression of KLF2-dependent genes and improves vascular remodeling. 54 Traf7 ECko embryos also displayed higher expression of immediate-early response genes, Ier3 and basic-helix-loop-helix transcription factor Bhlhe40, indicating once again embryonic distress resulting from insufficient oxidation in the absence of TRAF7. On the other hand, Klf4 was not among DEGs significantly upregulated in Traf7 ECko embryos compared to Traf7 EChet controls (Table S2), implying that the transcription of Klf4 is not regulated by TRAF7 in embryonic endothelial cells.
Conditional deletion of Traf7 in postnatal endothelium leads to brain hemorrhage Since global or endothelial-specific deletion of Traf7 resulted in embryonic lethality, we next examined whether TRAF7 expression is required for the integrity of postnatal endothelium. We crossed Traf7 fl/fl mice to Cdh5(PAC)-Cre ERT2 -trangenic animals, which demonstrate high levels of recombinase-mediated gene deletion in vascular endothelial cells following administration of tamoxifen. 55,56 Traf7 deletion was induced starting at postnatal day 0 (P0) by daily oral feeding of tamoxifen. Twelve days after the initiation of tamoxifen administration, we noted that one of TRAF7 fl/-$Cdh5(PAC)-CreERT2 (Traf7 iECko = inducible endothelial cell knockout) mice developed a pronounced ataxia with seizure-like behavior. At this point, we euthanized all experimental mice in this group and examined the brains. The gross inspection of whole brains demonstrated that, in contrast to Traf7 iEChet littermates, Traf7 iECko mice exhibited prominent focal hemorrhages ( Figure 5A), while the quantification revealed a significantly higher number of hemorrhagic foci in Traf7 iECko brains compared to the controls ( Figure 5B). A closer examination of neonatal brains confirmed bleeding in Traf7 iECko mice ( Figure 5C). Blood vessels in the brain and choroid plexus of the mutant animals were prone to rupture, releasing erythrocytes. The brain was the only affected organ in our experiments, probably due to extensive vascular remodeling of the brain during postnatal development. 57 The analysis of Klf2 and Klf4 in P7 brains revealed a significantly lower expression of Klf2, but not of Klf4, in Traf7 iECko brains compared to Traf7 iEChet littermates, mirroring the results obtained in Traf7-deficient embryos ( Figure 5D). Our finding that postnatal endothelial-specific deletion of Traf7 resulted in brain hemorrhage is in agreement with previously published results demonstrating that Mekk3 is required for the integrity of postnatal vasculature. 58

Association of TRAF7 with SCRIB
Although the role of the MEKK3-MEK5-ERK5-KLF2 pathway in blood flow-induced shear stress signaling in endothelial cells has been extensively studied over the years, 36 iScience Article TRAF7 to pull down associated proteins. In order to do this, the coding regions of full-length TRAF7 and meningioma mutant TRAF7 N520S were expressed as fusion proteins with FLAG epitope tags at the N terminus. Plasmids encoding FLAG-fusion proteins of bacterial alkaline phosphatase (BAP) and COP1 were used as negative controls. We transfected HEK293 cells with vectors expressing fusion proteins and precipitated associated proteins with anti-FLAG magnetic beads ( Figure S5A). Interestingly, beadbound TRAF7 and TRAF7 N520S formed high-molecular-weight complexes, which were partially resistant to solubilization in the reducing Laemmli buffer. Both TRAF7 fusion proteins associated with endogenous MEKK3, MEKK2, and MEK5, but not MARK2, another kinase that was also previously shown to interact with TRAF7. 12 There also was no ERK5 in TRAF7 precipitates, indicating that ERK5 may not be a part of the TRAF7 complex. Importantly, MEKK2, MEKK3, and MEK5 precipitated with neither BAP nor COP1, indicating specificity of binding to TRAF7. Next, we performed mass spectrometry analysis of TRAF7-interacting proteins precipitated from HEK293 cells. After subtracting non-specific proteins precipitated with BAP beads, SCRIB (also known as SCRB1), a homolog of Drosophila melanogaster SCRIBBLE, appeared to be the most abundant of TRAF7-interacting proteins ( Figure S5B). Western blot analysis revealed that SCRIB specifically precipitated with both TRAF7 and TRAF7 N520S , but not with BAP or COP1 ( Figure 6A). While coincidental association of TRAF7 with SCRIB was first mentioned in the original TRAF7-MEKK3 study, no results were shown. 12 More recently, SCRIB was also found as one of 79 proteins associated with TRAF7, and TRAF7 was identified as one of 95 proteins associated with SCRIB in human interactome iScience Article studies. 19 Taken together, these results demonstrate that TRAF7 interacts with SCRIB in addition to MEKK2, MEKK3, and MEK5.
To further characterize the association of MEKK3, MEK5, and SCRIB with TRAF7, we expressed truncated TRAF7 fragments and co-precipitated endogenous associated proteins ( Figures 6B and 6C). In agreement with previous reports, 12,14 the C-terminal WD40 domain (375-670) was required and sufficient for interaction of TRAF7 with MEKK3. Interestingly, MEK5 associated with two distinct regions in TRAF7. The first iScience Article TRAF7 region (121-292) comprises the RING finger and zinc-finger domains, and the second region (375-670) includes the WD40 domain, which also binds MEKK3. The shortest truncated mutant of TRAF7 able to bind SCRIB was TRAF7(1-292). Two shorter mutants, TRAF7(1-127) and TRAF7(121-292), did not associate with SCRIB, suggesting that a fragment consisting of the N-terminal Serine-Threonine (S/T)-rich RING finger and ZF of TRAF7 was necessary for interaction with SCRIB. Thus, there are two non-overlapping regions in TRAF7: the N-terminal region of TRAF7 that binds SCRIB and the C-terminal that binds MEKK3. Interestingly, MEK5 binds to both regions.
To examine whether TRAF7 directly interacts with SCRIB, we ectopically co-expressed both of them as fusion fluorescent proteins ( Figure S6). Full-size SCRIB was expressed as a fusion with EGFP, while fullsize TRAF7 and its fragments were expressed as fusion proteins with mCherry. When expressed alone, the majority of full-size TRAF7 was localized in a discrete vesicular pattern in the cytosol, as was previously shown. 12 In contrast, TRAF7(1-292) was diffusely distributed throughout the cytoplasm, while TRAF7(1-384) formed even bigger aggregates in the cytoplasm, suggesting that the CC domain of TRAF7 is important for self-interaction. When expressed alone, SCRIB-EGFP showed a diffuse intracellular distribution, but when expressed together with truncated TRAF7 fragments, it joined the TRAF7-containing aggregates, suggesting direct interaction of two proteins.

TRAF7 and SCRIB are essential for phosphorylation of ERK5 induced by fluid flow
As a key integrator of fluid shear stress signaling, ERK5 is required for the maintenance of blood vessel integrity and vascular homeostasis. 31,32,34,60 It is activated by fluid flow shear stress in endothelial cells following phosphorylation by MEK5. 61 To understand whether TRAF7 and SCRIB are important for phosphorylation of ERK5 in endothelial cells activated by fluid flow, we exposed a confluent monolayer of human umbilical vein endothelial cells (HUVECs) to pulsating shear stress on an orbital shaker. 62 Western blot analysis showed a notable upward shift in molecular weight of ERK5 in cells exposed to shear stress and transfected with negative control endoribonuclease-prepared small interfering RNAs (esiRNAs) against iScience Article Renilla luciferase (RLUC) and MEKK2 (Figure 7, lanes 2 and 10). This shift was consistent with expected activation and phosphorylation of ERK5, which is not mediated by MEKK2. 61,63 In contrast, silencing of TRAF7, SCRIB, and MEKK3 resulted in downregulated phosphorylation of ERK5 following exposure to shear stress, suggesting that TRAF7 and SCRIB together with MEKK3 may be important for phosphorylation of ERK5 induced by blood flow in endothelial cells. Because both KLF2 and KLF4 were shown to be downstream of increased MEKK3-MEK5-ERK5 signaling in cerebral cavernous malformations, 64,65 we next examined the expression of KLF2 and KLF4 in shear stress-exposed HUVECs. The results demonstrated an upregulation of KLF2 as well as KLF4 proteins in HUVEC transfected with negative control esiRNAs against RLUC and MEKK2 after exposure to shear stress (lanes 2 and 10 in Figure 7A). In contrast, there was a lower upregulation of these transcription factors in cells transfected with esiRNAs against TRAF7, SCRIB, or MEKK3 (lanes 4, 6 and 8 in Figure 7A), indicating that TRAF7 as well as SCRIB are important for upregulation of KLF2 and KLF4 in HUVEC in response to shear stress in vitro.

DISCUSSION
Blood flow through vessels exerts circulatory pressure and fluid shear stress on the endothelial cells that line the luminal surface. 66 The level of biomechanical stress varies from region to region within the vasculature and plays a critical role in shaping the vascular system to optimize perfusion. The blood flow-responsive endothelial transcription factor KLF2 is known to be activated by shear stress signaling mediated through the MEKK3-MEK5-ERK5 axis. 37 Indeed, differential Klf2 expression exhibited the highest statistical significance with substantial downregulation in global as well as endothelial-specific Traf7-deficient embryos compared to controls, suggesting that like MEKK3, MEK5, and ERK5, TRAF7 is necessary for Klf2 expression in endothelial cells. It is interesting, however, that Klf2-deficient embryos die almost two days later than mice lacking Traf7, Mekk3, Mek5, or Erk5, e.g., between E11.5 to E14.5 depending on the background. 40,42,67,68 Moreover, Klf2-deficient embryos die due to embryonic heart failure but not from anemia or structural vascular defects. These results suggest that genes other than Klf2 may be the cause of early death of Traf7 GLOko embryos. 42 One of those genes may be Klf4 because double-knockout Klf2 À/À ;KLF4 À/À embryos die by E10.5 due to disruptions of the endothelial cell layer of the primary head vein. 69 However, no significant difference was observed in the expression of Klf4 in neither Traf7 GLOko nor Traf7 ECko embryos compared to their controls (Tables S1 and S2), indicating that other genes may also contribute to impaired endothelial integrity in Traf7 GLOko embryos.
Although the molecular mechanism of shear stress sensing in endothelial cells has been extensively studied, much still remains to be investigated due to the complexity of blood flow through the vascular network, including variations in intensity, direction, and pulsatility. Here, we demonstrate that in addition to MEKK3 and MEK5, TRAF7 binds to a planar cell polarity protein, SCRIB, which was originally identified in Drosophila as a large cytoplasmic protein containing 16 leucine-rich repeats (LRR), two leukemia-associated-protein (LAP)-specific domains, and four PSD95/Dlg1/zo-1 (PDZ) domains. 70 In Drosophila, it associates with discs-large (DLG), and lethal-giant larvae (LGL) proteins, while in mammals SCRIB binds numerous partners and functions as a master scaffold in planar cell polarity, adhesion, synaptogenesis, and proliferation processes. 71 In cultured mammalian epithelial cells, SCRIB localizes to the basolateral membrane and appears to overlap with adherens junctions. 72 It plays important roles in cell adhesion, planar polarity, and asymmetric divisions. 73 There are conflicting reports regarding the role of SCRIB in endothelial cells. Although the functional consequences of these interactions remain unknown, SCRIB was shown to bind non-filamentous vimentin near membrane borders between cells in confluent HUVEC cultures. 74 In another study, SCRIB interacted with integrin a5 in HUVECs and was shown to be important for directed migration and angiogenesis of intersegmental vessels in zebrafish embryos. 75 In mice, endothelial-specific deletion of Scrib significantly impairs LPS-induced vascular cell adhesion protein 1 (VCAM1) expression and leukocyte adhesion in response to TNF treatment, suggesting that SCRIB facilitates endothelial inflammatory signaling. 76 On the other hand, deletion of Scrib in ApoE-deficient mice impairs vascular permeability and promotes atherosclerosis development, indicating that SCRIB contributes to anti-inflammatory responses in endothelial cells. 77 The latter observation is consistent with the inward arterial remodeling and accelerated atherosclerosis seen in Mekk3-deficient mice. 78 These results imply that SCRIB and MEKK3 may have an overlapping function, which supports our finding that SCRIB knockdown reduced shear stress-induced phosphorylation of ERK5 in HUVECs. iScience Article Human Scrib mutations are associated with the most severe type of neural tube closure defects, including craniorachischisis and spina bifida. 79,80 Interestingly, the circletail mouse (Scrib Crc ) closely mirrors the phenotypic defects described in humans. 81,82 In Scrib Crc mice, a spontaneous mutation due to a single base insertion after 3182 bp of coding sequence results in frameshift at codon 947 and truncation of SCRIB protein after the second PDZ domain, leaving intact exons for all LLRs, two LAP domains, and the first two PDZ domains. Nevertheless, Scrib Crc embryos failed to initiate neural tube closure at E8.5 and exhibited an open neural tube from the midbrain/hindbrain boundary throughout the spine. 83,84 Likewise, whole-body Scrib-knockout mice (Scrib Gko ) die prenatally and display an array of developmental abnormalities, including neural tube closure defects and cochlear hair cell orientation defects. 85 In these mice, the authors excised exons 4-12, encoding LLRs 4-16, but kept in place the coding sequence for LLRs 1-3, two LAP domains, and all PDZ domains. This may explain why the phenotype of Scrib Crc and Scrib Gko mice is very different from the phenotype of Traf7 GLOko mice, which had no obvious neural tube closure defects and died around E10 from failed blood vessel integrity. Thus, we cannot exclude that SCRIB might play a role in shear stress-dependent TRAF7-MEKK3-MEK5-ERK5 axis in endothelial cells during embryonic development because in both Scrib-knockout mice the majority of the SCRIB coding sequence was left intact in the genome. These leftover fragments might still bind TRAF7 and potentially play a role in TRAF7-dependent shear stress signaling in endothelial cells. Future studies are needed to assess whether SCRIB participates in the sensing of blood flow physical forces by the TRAF7-MEKK3-MEK5 complex during embryogenesis.
When TRAF7 was discovered as a MEKK3 synergistic partner in activation of NF-kB, p38, and c-June N-terminal kinase (JNK), it was demonstrated that TRAF7 is an E3 ubiquitin ligase capable of self-ubiquitination, 12 although E3 ubiquitin ligase activity was not important for signal transduction in both original studies. 12,14 Subsequently, TRAF7 was shown to ubiquitinate a number of molecules, including c-MYB, 16 MYOD1, 17 NEMO, 18 FLICE-like inhibitory protein (c-FLIP L , also known as CFLAR), 86 p53, 87 and KLF4. 88 In every study ubiquitination resulted in degradation of the substrate and activation of pro-inflammatory and/or pro-apoptotic pathways, such as NF-kB, or MAP kinases. 7 For example, it was shown that TRAF7 associated with KLF4 through its ZF-CC region and was responsible for ubiquitination and proteasomedependent degradation of KLF4 through its RING-dependent E3 ubiquitin ligase activity to promote hepatocellular carcinoma progression. 88 In contrast to the above observations, we found that TRAF7 is a part of blood flow-induced shear stress-responsive MEKK3-MEK5-ERK5 signaling pathway, which plays an anti-inflammatory role in the endothelium. 37 Moreover, we did not detect any significant differences in the expression of MEKK3 or MEK5 in Traf7 GLOko embryos compared to WT controls ( Figure S1D). Furthermore, no significant changes in the expression of MEKK3, MEK5, or SCRIB were observed in the lysates of HEK293 cells ectopically expressing full size or fragments of TRAF7 ( Figure 6C) or in TRAF7-deficient HUVECs exposed to shear stress ( Figure 7). We also notice no increases in molecular weight of TRAF7-associated proteins, except SCRIB ( Figure 6A), following association with TRAF7. The investigation whether higher-molecular-weight variants of SCRIB are the results of protein aggregation or ubiquitination would be an interesting subject of future investigations.

Limitations of the study
In this study, we generated Traf7-deficient mice to elucidate the physiological role of TRAF7 and describe phenotypes resulted from global and endothelial cell-specific deletion of Traf7. Both Traf7 GLOko -and Tra-f7 ECko -deficient embryos failed to develop beyond day 10 of embryonic development due to impaired blood vessel integrity linking TRAF7 with MEKK3-MEK5-ERK5 signaling pathway. One possible mechanism through which TRAF7 modulates shear stress signaling is by association with SCRIB, a planar cell polarity protein. We show that SCRIB is important for shear stress-induced phosphorylation of ERK5 in HUVECs.
Transfections of HUVECs with esiRNA worked consistently well, but plasmid transfections into HUVECs proved to be difficult and inefficient in our hands as it was shown before for HUVECs. 89 This presented an obstacle for TRAF7 interaction studies in endothelial cells. Therefore, we performed all in vitro TRAF7 interaction studies in HEK293 cells. Major unanswered questions include how TRAF7 activates MEKK3-MEK5-ERK5 signaling, does it function as E3 ligase for possible ubiquitination of these associated proteins, as well as the molecular mechanism of endothelium weakness in TRAF7-deficient animals. These and other questions are a matter for future studies.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
The authors thank Anthony C. Johnson for editing the manuscript and Hannah Homburg, Alec Delsigne, and Cynthia Bulmer for technical assistance.

DECLARATION OF INTERESTS
The authors declare no competing interests.

INCLUSION AND DIVERSITY
One or more of the authors of this paper self-identifies as a member of the LGBTQIA+ community.